Human blue-light photoreceptor hCRY2

ABSTRACT

The present invention relates to a novel member of the blue-light photoreceptor family of receptors. In particular, isolated nucleic acid molecules are provided encoding the human hCRY2 receptor. hCRY2 polypeptides are also provided as are vectors, host cells, antibodies, and recombinant methods for producing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/105,254, filed Jun. 26, 1998, which is a divisional of U.S.application Ser. No. 08/964,268, filed Nov. 4, 1997 (now U.S. Pat. No.6,114,503), which claims benefit under 35 §U.S.C. 119(e) of U.S.Provisional Application No. 60/030,189, filed Nov. 4, 1996. Each of theabove referenced patents and patent applications is hereby incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a novel human blue-lightphotoreceptor. More specifically, isolated nucleic acid molecules areprovided encoding a human blue-light photoreceptor. Human blue-lightphotoreceptor polypeptides are also provided, as are vectors, hostcells, antibodies, and recombinant methods for producing the same.

[0004] 2. Related Art

[0005] In many organisms, the photolyase/photoreceptor family ofproteins mediates DNA repair. In plants, certain developmental processesare regulated by blue-light. This regulation occurs by a photoinducedelectron transfer reaction (Taylor, J. S., Acc. Chem. Res. 27:76-82(1994); Menkens, A. E. et al., Biochemistry 34:6892-6899 (1995); Heelis,P. F. et al., Photochem. Photobiol. 95:89-98 (1996); and Sancar A.,Science 272:48-49 (1996)). Indeed, to date, most of the work concerningblue-light photoreceptors has been conducted in plants (Cashmore, A. R.et al., International Patent Application WO 96/01897 (1996); Hinnemann,H., Photochem. Photobol. 61:22-31(1995); Short, T. W. et al., Annu. Rev.Plant. Physiol. Plant Mol. Biol. 45:143-171 (1994); Hohl, N. et al.,Photochem. Photobiol. 55:239-245 (1992)) and fungi (Dunlap, J. C., Annu.Rev. Physiol. 55:683-728 (1993)). In plants, blue-light inducesresponses such as photomorphogenesis, phototropism and hypocotylelongation. In particular, it has been demonstrated that the HY4 gene ofA. thaliana, which encodes the CRY1 protein, is required for blue-lightinduced hypocotyl elongation (Ahmad, M., et al., Nature 366:162-166(1993)).

[0006] In animals, most of the work on light response (other thanvision) has been concentrated on circadian clocks. In D. melanogaster,two genes have been cloned, timeless and period, which regulate thecircadian rhythm (Myers, M. P. et al., Science 270:805-808 (1995);Gekakis, N. et al., Science 270:811-814 (1995)). Both appear to betranscription factors for which activity is regulated by light. Amutation in the golden hamster tau gene disrupts the circadian clock(Ralph and Menaker, 1988). Three mouse genes, CLOCK, ICER, and CREM,which are involved in the control of circadian rhythm, have beeninvestigated in some detail (Vitaterna et al., M. H. et al., Science264:719-725 (1994); Sassone-Corsi, P. A., Rev. Cell Dev. Biol.11:355-377 (1995); Foulkes, N. S. et al., Nature 381:83-85 (1996)). Eachof these three gene products appears to be a transcriptional repressorfor which activity is regulated by light. However, how the light signalis transmitted to these transcriptional regulators is not known.

[0007] Currently, the photolyase/photoreceptor protein family is knownto contain three members: the cyclobutane pyrimidine dimer (Pyr<>Pyr)photolyase (photolyase), the (6-4) photolyase, and the blue-lightphotoreceptor (Todo, T. et al., Science 272:109-112 (1996)). The genefor the classical Pyr<>Pyrphotolyase has been cloned and the enzyme hasbeen purified from many organisms, including Escherichia coli,Saccharomyces cerevisiae, Drosophila melanogaster, and Carassius auratus(Sancar, A., Mutation Res. 236:147-160(1990); Kato, T. et al., Nucl.Acids Res. 22:4119-4124 (1994); and Yasui, A. et al., EMBO J.13:6143-6151 (1994). The (6-4) photolyase has been found in D.melanogaster (Todo, T. et al., Nature 361:371-374 (1993); Kim, S. T. etal., J. Biol. Chem. 269:8535-8540 (1994)), Xenopus laevis, and Crotalusatrox (Kim, S. T. et al., Photochem. Photobiol. 63:292-295 (1996)).

[0008] Concerning the cloning of (6-4) photolyase genes, only theDrosophila gene has been cloned and sequenced (Todo, T. et al., Science272:109-112 (1996)). The genes for the apoproteins of the blue-lightphotoreceptors of Arabidopsis thaliana (Ahmad, M., Nature 366:162-166(1993)), Sinapis alba (Batschauer, A., Plant J. 4:705-709 (1993);Malhotra, K. et al., Biochemistry 34:6892-6899 (1995)), andChlamydomonas reinhardtii (Small, G. D., et al., Plant Molec. Biol.28:433-454 (1995)) have been cloned and sequenced. The photoreceptors ofA. thaliana (Malhotra, K. et al., Biochemistry 34:6892-6899 (1995); Lin,C. et al., Science 269: 968-970 (1995)) and S. alba (Malhotra, K. etal., Biochemistry 34:6892-6899 (1995)) have been purified andcharacterized.

[0009] Circadian regulation of human and animal physiology, andparticularly circadian regulation mediated by blue-light photoreceptors,is poorly understood. Thus, there is a need for an isolated humanblue-light photoreceptor gene, the polypeptide encoded by that gene, andantibodies specific for that polypeptide.

SUMMARY OF THE INVENTION

[0010] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the human blue-light photoreceptorhCRY2 [hereinafter “hCRY2”] receptor having the amino acid sequenceshown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNAclone deposited in a bacterial host as ATCC Deposit Number 97769 on Oct.22, 1996.

[0011] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention, tohost cells containing the recombinant vectors, to host cells containingan isolated polypeptide, as well as to methods of making such vectorsand host cells and for using them for production of hCRY2 polypeptidesor peptides by recombinant techniques.

[0012] The invention further provides an isolated hCRY2 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0013] The invention further provides isolated antibodies that bindspecifically to the full length hCRY2 receptor, the mature hCRY2receptor, the hCRY2 receptor extracellular domain, the hCRY2 receptortransmembrane domain, the hCRY2 receptor intracellular domain, andepitope-bearing portions of the hCRY2 receptor.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGS. 1A-1E show the nucleotide (SEQ ID NO:1) and deduced aminoacid (SEQ ID NO:2) sequences of hCRY2 receptor. The protein has apredicted leader sequence of about 22 amino acid residues (underlined)and a deduced molecular weight of about 81 kDa. It is further predictedthat amino acid residues from about 23 to about 514 (about 1 to about492 in SEQ ID NO:2) constitute the extracellular domain; from about 515to about 527 (about 493 to about 505 in SEQ ID NO:2) the transmembranedomain; and from about 528 to about 593 (about 506 to about 571 in SEQID NO:2) the intracellular domain.

[0015]FIG. 2 shows the regions of similarity between the amino acidsequences of the hCRY2 receptor protein and hCRY1 (SEQ ID NO:3).

[0016]FIG. 3 shows an analysis of the hCRY2 receptor amino acidsequence. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown. In the “Antigenic Index—Jameson-Wolf”graph, amino acid residues about 30 to about 39, about 45 to about 52,about 77 to about 92, about 96 to about 103, about 158 to about 169,about 178 to about 187, about 253 to about 261, about 293 to about 301,about 320 to about 331, about 338 to about 346, about 350 to about 356,about 437 to about 448, and about 534 to about 541 in FIG. 1 correspondto the shown highly antigenic regions of the hCRY2 receptor protein.These highly antigenic fragments in FIG. 1 correspond to the followingfragments, respectively, in SEQ ID NO:2: amino acid about 8 to about 17,about 23 to about 30, about 55 to about 70, about 74 to about 81, about136 to about 147, about 156 to about 165, about 231 to about 239, about271 to about 279, about 298 to about 309, about 316 to about 324, about328 to about 334, about 415 to about 426, and about 512 to about 519.

[0017] FIGS. 4A-4C shows the sequence comparison of E. coli photolyase(E.c.) (SEQ ID NO:4), Arabidopsis HY4 photoreceptor (A.t.) (SEQ IDNO:5), Drosophila melanogaster (6-4) photolyase (D.m.) (SEQ ID NO:6),and the human blue-light photoreceptors hCRY1 (SEQ ID NO:3) and hCRY2(SEQ ID NO:2). Amino acid residues which are identical in the entire setare boxed.

[0018]FIG. 5 shows the maps of plasmids pDH1996-1 and pDH1996-2, whichwere used to express hCRY1 and hCRY2, respectively, as maltose bindingfusion proteins. PDH1996-1 contains the hCRY1 cDNA. pDH1996-2 containsthe hCRY2 cDNA. The arrows indicate the length and direction oftranscription of the maltose binding protein (malE), blal, andphotoreceptor genes.

[0019] FIGS. 6A-6C shows the absorption and fluorescence spectra ofhCRY1 and hCRY2 MBP fusion proteins. The dashed line represents thespectra of hCRY1 and the solid line represents the spectra of hCRY2. (A)Absorption spectra. (B) Fluorescence excitation and emission spectra ofthe hCRY1 and hCRY2 chromophores at pH 2. Fluorescence excitationspectra were recorded by monitoring emission at 520 nm. Fluorescenceemission spectra were recorded by using excitation at 450 nm. (C)Fluorescence excitation and emission spectra of hCRY1 and hCRY2chromophores at pH 10. Fluorescence excitation spectra were recorded bymonitoring emission at 470 nm. Fluorescence emission spectra wererecorded by using 380 nm excitation.

DETAILED DESCRIPTION

[0020] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a hCRY2 polypeptide having theamino acid sequence shown in SEQ ID NO:2. The amino acid sequence wasdeduced from the sequence of a cloned hCRY2 cDNA. The sequenced cDNAclone was obtained using RACE-PCR (infra). The hCRY2 protein of thepresent invention shares sequence homology with hCRY1 (FIG. 2; SEQ IDNO:3).

[0021] A cDNA encoding a maltose binding protein-hCRY2 fusion protein,including amino acid residues −15 to 571 (SEQ ID NO:2) was deposited onOct. 22, 1996 at the American Type Culture Collection, PatentDepository, 10801 University Boulevard, Manassas, Va. 20110-2209, andgiven accession number 97769. The hCRY2 sequence is contained betweenthe EcoR V and Hind III sites in the polylinker of the Mal-C2 vector(NEB, Beverly, Mass.).

[0022] Nucleic Acid Molecules

[0023] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0024] Using the information provided herein, such as the nucleotidesequence in SEQ ID NO:1, a nucleic acid molecule of the presentinvention encoding a hCRY2 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in SEQ ID NO:1 was discovered in a cDNA libraryderived from human fetal brain. The hCRY2 gene was also identified incDNA libraries from the following tissues: synovial sarcoma, restingT-cell, infant brain, cerebellum, endometrial tumor, testes tumor, adultretina, chondrosarcoma, breast, and pituitary.

[0025] The determined nucleotide sequence of the hCRY2 cDNA of SEQ IDNO:1 contains an open reading frame encoding a protein of about 593amino acid residues, with a predicted leader sequence of about 22 aminoacid residues, and a deduced molecular weight of about 81 kDa. The hCRY2protein shown in SEQ ID NO:2 is about 74% identical and about 85%similar to hCRY1 (FIG. 2; SEQ ID NO:3).

[0026] As indicated, the present invention also provides the matureform(s) of the hCRY2 receptor of the present invention. According to thesignal hypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature hCRY2 polypeptides having the amino acid sequence encoded bythe cDNA clone contained in the host identified as ATCC Deposit No.97769and as shown in SEQ ID NO:2. By the mature hCRY2 protein having theamino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC Deposit 97769 is meant the mature form(s) of thehCRY2 receptor produced by expression in a mammalian cell (e.g., COScells, as described below) of the open reading frame encoded by thehuman DNA sequence of the clone contained in the vector in the depositedhost. This clone lacks amino acid residues −22 to −16 in SEQ ID NO:2. Asindicated below, the mature hCRY2 receptor having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No.97769may or may not differ from the predicted “mature” hCRY2 protein shown inSEQ ID NO:2 (amino acids from about 1 to about 571) depending on theaccuracy of the predicted cleavage site.

[0027] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are available.For instance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) andvon Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0028] In the present case, the predicted amino acid sequence of thecomplete hCRY2 polypeptide of the present invention was analyzed by acomputer program (“PSORT”) (K. Nakai and M. Kanehisa, Genomics14:897-911 (1992)), which is an expert system for predicting thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis by the PSORT programpredicted the cleavage sites between amino acids 27 and 28 in SEQ IDNO:2. However, based on homology to the hCRY1 protein (FIG. 2; SEQ IDNO:3), the cleavage site is predicted to be between amino acids −1 and 1in SEQ ID NO:2. Thus, the leader sequence for the hCRY2 protein ispredicted to consist of amino acid residues −1 to −22 in SEQ ID NO:2,while the mature hCRY2 protein is predicted to consist of amino acidsresidues 1-571 in SEQ ID NO:2.

[0029] As one of ordinary skill would appreciate, however, due to thepossibilities of sequencing errors, as well as the variability ofcleavage sites for leaders in different known proteins, the full-lengthhCRY2 polypeptide comprises about 593 amino acids, but may be anywherein the range of about 580 to about 600 amino acids; and the leadersequence is about 22 amino acids, but may be anywhere in the range ofabout 15 to about 55 amino acids.

[0030] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0031] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0032] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising the open reading frame (ORF) shown in SEQ IDNO: 1; DNA molecules comprising the coding sequence for the mature hCRY2receptor shown in SEQ ID NO:2; and DNA molecules which comprise asequence substantially different from those described above but which,due to the degeneracy of the genetic code, still encode the hCRY2receptor. Of course, the genetic code is well known in the art. Thus, itwould be routine for one skilled in the art to generate such degeneratevariants.

[0033] In another aspect, the invention provides isolated nucleic acidmolecules encoding the hCRY2 polypeptide having an amino acid sequenceencoded by the cDNA set forth in SEQ ID NO:1 and by the clone containedin the plasmid deposited as ATCC Deposit No. 97769 on Oct. 22, 1996. Infurther embodiments, this nucleic acid molecule will encode the maturepolypeptide or the full-length polypeptide lacking the N-terminalmethionine. The invention further provides an isolated nucleic acidmolecule having the nucleotide sequence shown in SEQ ID NO:1 or thenucleotide sequence of the hCRY2 receptor cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the hCRY2 receptor gene in human tissue, for instance, byNorthern blot analysis.

[0034] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in SEQ ID NO:1 isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or 1750 nt inlength are also useful according to the present invention, as arefragments corresponding to most, if not all, of the nucleotide sequenceof the deposited cDNA or as shown in SEQ ID NO: 1. By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1.

[0035] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the hCRY2receptor extracellular domain (predicted to constitute amino acidresidues from about 1 to about 492 in SEQ ID NO:2); a polypeptidecomprising the hCRY2 receptor transmembrane domain (predicted toconstitute amino acid residues from about 493 to about 505 in SEQ IDNO:2); a polypeptide comprising the hCRY2 receptor intracellular domain(predicted to constitute amino acid residues from about 506 to about 571in SEQ ID NO:2); and a polypeptide comprising the hCRY2 receptorextracellular and intracellular domains with all or part of thetransmembrane domain deleted. As above with the leader sequence, theamino acid residues constituting the hCRY2 receptor extracellular,transmembrane and intracellular domains have been predicted by computeranalysis. Thus, as one of ordinary skill would appreciate, the aminoacid residues constituting these domains may vary slightly (e.g., byabout 1 to about 15 amino acid residues) depending on the criteria usedto define each domain.

[0036] Preferred nucleic acid fragments of the present invention alsoinclude nucleic acid molecules encoding epitope-bearing portions of thehCRY2 receptor protein. In particular, such nucleic acid fragments ofthe present invention include nucleic acid molecules encoding: apolypeptide comprising amino acid residues from about 8 to about 17 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 23to about 30 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 55 to about 70 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 74 to about 81 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 136 to about 147 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 156 toabout 165 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 231 to about 239 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 271 to about 279 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 298 to about 309in SEQ ID NO:2; a polypeptide comprising amino acid residues from about316 to about 324 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 328 to about 334 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 415 to about 426 in SEQ IDNO:2; and a polypeptide comprising amino acid residues from about 512 toabout 519 in SEQ ID NO:2. It is believed that the above polypeptidefragments are antigenic regions of the hCRY2 receptor. Methods fordetermining other such epitope-bearing portions of the hCRY2 protein aredescribed in detail below.

[0037] In addition, the present inventors have identified nucleic acidmolecules having nucleotide sequences related to extensive portion ofSEQ ID NO:1 which have been determined from the following related cDNAclones: HFCAD18R (SEQ ID NO:17); HDPFZ96R (SEQ ID NO:18); HBNAG83R (SEQID NO:19); and HJBAZ81R (SEQ ID NO:20). HFCAD18R (SEQ ID NO: 17) isrelated to nucleotides 1372 to 1661 of SEQ ID NO:1. HDPFZ96R (SEQ IDNO:18) is related to nucleotides 611 to 712 of SEQ ID NO:1. HBNAG83R(SEQ ID NO:19) is related to nucleotides 3546 to 3691 of SEQ ID NO:1.HJBAZ81R (SEQ ID NO:20) is related to nucleotides 2460 to 2600 of SEQ IDNO: 1.

[0038] The sequence of a public EST, having GenBank Accession No.AA338421, related to a portion of SEQ ID NO:1 is shown in SEQ ID NO:21.This public EST contains a region of 290 nucleotides that are related tonucleotides 1260-1549 of SEQ ID NO:1.

[0039] The sequence of another public EST, having GenBank Accession No.AA297444, related to a portion of SEQ ID NO: 1 is shown in SEQ ID NO:22.This public EST contains a region of 210 nucleotides that are related tonucleotides 1102-1311 of SEQ ID NO:1.

[0040] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the full-length cDNA set forth in SEQ ID NO:1 or the cDNA clonecontained in ATCC Deposit 97769. By “stringent hybridization conditions”is intended overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20g/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C.

[0041] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0042] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1).

[0043] Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3¢ terminal poly(A) tract of the hCRY2 receptorcDNA shown in SEQ ID NO:1), or to a complementary stretch of T (or U)resides, would not be included in a polynucleotide of the invention usedto hybridize to a portion of a nucleic acid of the invention, since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0044] As indicated, nucleic acid molecules of the present inventionwhich encode a hCRY2 polypeptide may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 22 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5¢ and 3¢ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the hCRY2receptor fused to the maltose binding protein sequence (see Examples 1and 2), or to Fc at the N- or C-terminus.

[0045] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the hCRY2 receptor. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0046] Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the hCRY2receptor or portions thereof. Also especially preferred in this regardare conservative substitutions.

[0047] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 95% identical, and more preferably at least 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotidesequence encoding the polypeptide having the complete amino acidsequence in SEQ ID NO:2 except for the N-terminal methionine (amino acidresidues −21 to 571 in SEQ ID NO:2); (c) a nucleotide sequence encodingthe polypeptide having the amino acid sequence at positions from about 1to about 571 in SEQ ID NO:2; (d) a nucleotide sequence encoding thepolypeptide having the amino acid sequence at positions from about 191to about 571 in SEQ ID NO:2; (e) a nucleotide sequence encoding thehCRY2 polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97769; (f) a nucleotide sequenceencoding the mature hCRY2 receptor having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97769; (g) anucleotide sequence encoding the hCRY2 receptor extracellular domain;(h) a nucleotide sequence encoding the hCRY2 receptor transmembranedomain; (i) a nucleotide sequence encoding the hCRY2 receptorintracellular domain; (j) a nucleotide sequence encoding the hCRY2receptor extracellular and intracellular domains with all or part of thetransmembrane domain deleted; and (k) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), (i), or (j).

[0048] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding ahCRY2 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the hCRY2receptor. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mutations of the reference sequence mayoccur at the 5¢ or 3¢ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.

[0049] As a practical matter, whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in SEQ ID NO:1 or to thenucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0050] The present application is directed to nucleic acid molecules atleast 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in SEQ ID NO: 1 or to the nucleic acid sequence of the depositedcDNA, irrespective of whether they encode a polypeptide having hCRY2receptor activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having hCRY2 receptor activity,one of skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having hCRY2 receptoractivity include, inter alia, (1) isolating the hCRY2 receptor gene orallelic variants thereof in a cDNA library; (2) in situ hybridization(e.g., “FISH”) to metaphase chromosomal spreads to provide precisechromosomal location of the hCRY2 receptor gene, as described in Vermaet al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and (3) Northern Blot analysis for detecting hCRY2receptor mRNA expression in specific tissues.

[0051] Preferred, however, are nucleic acid molecules having sequencesat least 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in SEQ ID NO:1 or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having hCRY2receptor activity.

[0052] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in SEQ ID NO: 1 will encode apolypeptide “having hCRY2 receptor activity.” It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving hCRY2 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

[0053] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0054] Vectors and Host Cells

[0055] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof hCRY2 polypeptides or fragments thereof by recombinant techniques.

[0056] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0057] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

[0058] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate heterologoushosts include, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS and Bowes melanoma cells; and plantcells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0059] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0060] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0061] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFe part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Feportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5—has been fused with Fe portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. See, D. Bennett etal., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K.Johanson et al., The Journal of Biological Chemistry, Vol. 270, No.16:9459-9471 (1995).

[0062] The hCRY2 receptor can be recovered and purified from recombinantcell cultures by well-known methods, including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

[0063] hCRY2 Polypeptides and Fragments

[0064] The invention further provides an isolated hCRY2 polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in SEQ ID NO:2, or a peptide or polypeptidecomprising a portion of the above polypeptides.

[0065] It will be recognized in the art that some amino acid sequencesof the hCRY2 receptor can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

[0066] Thus, the invention further includes variations of the hCRY2receptor which show substantial hCRY2 receptor activity or which includeregions of hCRY2 protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. As indicated above, guidance concerning which aminoacid changes are likely to be phenotypically silent can be found inBowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

[0067] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

[0068] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the hCRY2 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

[0069] The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al. (Nature 361:266-268(1993)) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, thehCRY2 receptor of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation.

[0070] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0071] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of substitutions for any givenhCRY2 polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3.

[0072] Amino acids in the hCRY2 protein of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

[0073] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced orcontained in a recombinant host cell is considered “isolated” for thepurposes of the present invention. Also intended as “isolated” is apolypeptide that has been purified, partially or substantially, from arecombinant host or a native source. For example, a recombinantlyproduced version of the hCRY2 receptor can be substantially purified bythe one-step method described in Smith and Johnson, Gene 67:31-40(1988).

[0074] The polypeptides of the present invention also include thecomplete polypeptide encoded by the deposited cDNA; the maturepolypeptide encoded by the deposited the cDNA; amino acid residues −22to 571 of SEQ ID NO:2; amino acid residues −21 to 571 of SEQ ID NO:2;amino acid residues 1 to 571 of SEQ ID NO:2; amino acid residues 191 to571 of SEQ ID NO:2; the extracellular domain; the transmembrane domain;and the intracellular domain, as well as polypeptides which are at least95% identical, more preferably at least 96%,97%, 98% or 99% identical tothe polypeptides described above, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

[0075] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a hCRY2polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the hCRY2 receptor. In otherwords, to obtain a polypeptide having an amino acid sequence at least95% identical to a reference amino acid sequence, up to 5% of the aminoacid residues in the reference sequence may be deleted or substitutedwith another amino acid, or a number of amino acids up to 5% of thetotal amino acid residues in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy-terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

[0076] As a practical matter, whether any particular polypeptide is atleast 95%, 96%, 97%, 98% or 99% identical to those described above canbe determined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

[0077] The polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0078] It is believed that the hCRY2 receptor is involved in thecircadian regulation of mammalian physiology. Thus, ligands which bindto the hCRY2 receptor would be useful in treating patients with primarysleep disorders (e.g., disorders with no apparent cause). Ligands whichbind to the hCRY2 receptor would also be useful in treating patientswith sleep disorders caused by odd working hours (e.g., among patientswho work during the night or who work rotating shifts).

[0079] It is also believed that the hCRY2 receptor is involved inmediating repair of damage to DNA, proteins, cells or tissue (e.g.,skin) caused by ultraviolet light. Thus, ligands which bind to the hCRY2receptor would be useful in treating patients suffering from UV damage.

[0080] As indicated below, the hCRY2 polypeptides of the presentinvention can be used to generate antibodies. Such antibodies can beused to investigate the expression, regulation, and ligand bindingproperties of the hCRY2 receptor.

[0081] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide described herein. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0082] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A., Antibodies that reactwith predetermined sites on protein, Science 219:660-666 (1983).Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals.

[0083] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

[0084] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between at least about 15to about 30 amino acids contained within the amino acid sequence of apolypeptide of the invention.

[0085] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate hCRY2 receptor-specific antibodies include: apolypeptide comprising amino acid residues from about 8 to about 17 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 23to about 30 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 55 to about 70 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 74 to about 81 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 136 to about 147 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 156 toabout 165 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 231 to about 239 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 271 to about 279 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 298 to about 309in SEQ ID NO:2; a polypeptide comprising amino acid residues from about316 to about 324 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 328 to about 334 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 415 to about 426 in SEQ IDNO:2; and a polypeptide comprising amino acid residues from about 512 toabout 519 in SEQ ID NO:2. As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe hCRY2 receptor protein.

[0086] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. Houghten, R. A., Generalmethod for the rapid solid-phase synthesis of large numbers of peptides:Specificity of antigen-antibody interaction at the level of individualamino acids, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0087] As one of skill in the art will appreciate, hCRY2 polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric hCRY2 protein or proteinfragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).

[0088] Detection of hCRY2 Gene Expression

[0089] The expression level of the hCRY2 gene can be readily assayed byone of ordinary skill in the art. By “assaying the expression level ofthe gene encoding the hCRY2 protein” is intended qualitatively orquantitatively measuring or estimating the level of the hCRY2 protein orthe level of the mRNA encoding the hCRY2 receptor in a biological sample(e.g., by determining or estimating absolute protein level or mRNAlevel).

[0090] By “biological sample” is intended any biological sample obtainedfrom an individual, cell line, tissue culture, or other source whichcontains hCRY2 protein or mRNA. Such tissues include cerebellum, retina,breast, pituitary, heart, placenta, lung, skeletal muscle, kidney, andpancreas. Biological samples include mammalian tissues which containhCRY2 protein. Preferred mammals include monkeys, apes, cats, dogs,cows, pigs, horses, rabbits, and humans. Particularly preferred arehumans.

[0091] Total cellular RNA can be isolated from a biological sample usingthe single-step guanidinium-thiocyanate-phenol-chloroform methoddescribed in Chomezynski and Sacchi (Anal. Biochem. 162:156-159 (1987)).Levels of mRNA encoding the hCRY2 receptor are then assayed using anyappropriate method. These include Northern blot analysis (Harada et al.,Cell 63:303-312 (1990)), S1 nuclease mapping (Harada et al., Cell63:303-312 (1990)), the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction (RT-PCR)(Fujita et al., Cell 49:35-36 (1990)), and reverse transcription incombination with the ligase chain reaction (RT-LCR).

[0092] As discussed supra, assaying hCRY2 protein levels in a biologicalsample can occur using antibody-based techniques. For example, hCRY2protein expression in tissues can be studied with classicalimmunohistological methods (Jalkanen, M. et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096(1987)). Other antibody-based methods useful for detecting hCRY2receptor gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitablelabels are known in the art and include enzyme labels, such as glucoseoxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc),and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0093] Chromosome Assays

[0094] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0095] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a hCRY2 receptor gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

[0096] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3¢ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes.

[0097] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

[0098] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance In Man, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0099] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0100] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1 Cloning the Human Photoreceptor

[0101] The cDNA clone (R1931) for the human photolyase homolog (Adams,M. D. et al., Nature 377:3-174 (1995)), carrying the 3′ terminal 1038 bpof the open reading frame gene, was obtained from R. K. Wilson(Washington University, St. Louis). The 5′ terminal part of the gene wasobtained using the 5′ RACE System for Rapid Amplification of cDNA ends(GibcoBRL, Gaithersburg, Md., USA) as described by the manufacturer andusing mRNA from the T093 human fibroblast cell line. The amplifiedproduct was digested with NcoI and Hind III and cloned into the NcoI/Hind III sites of the baculovirus expression vector p2Bac (Invitrogen,San Diego, Calif., USA) and the E. coli expression vector pKK233-2(Pharmacia, Uppsala, Sweden). Sequence of the gene was confirmed bydouble strand DNA sequencing using the Sequenase DNA sequencing kit (USBiochemical, Arlington, Ill., USA) and was in complete agreement withthe previously published sequence (Todo, T. et al., Science 272:109-112(1996)). A maltose binding protein (MBP) fusion construct was made byinserting the Bgl II/Hind III fragment carrying the entire photolyasehomolog coding region into the H I/Hind III site of the MBP expressionvector pMal-c2 (NEB, Beverly, Mass., USA). This construct was namedpDH1996-1.

[0102] The sequence of the second homolog was first identified bysearching a database containing approximately 1 million human ESTs,which was generated using high throughput automated DNA sequenceanalysis of randomly selected human cDNA clones (Adams, M. D. et al.,Nature 377:3-174 (1995); Adams, M. D. et al., Nature 355:632-634 (1992);and Adams, M. D. et al., Science 252:1651-1656 (1991)). Sequencehomology comparisons of each EST were performed against the GenBankdatabase using the blastn and tblastn algorithms (Altschul, S. F. etal., J. Mol. Biol. 215:403-410 (1990)). A specific homology search usingthe known human photolyase homolog 1 amino acid sequence against thishuman EST database revealed two ESTs (HGS6392 and HGS47815). Both werefrom a human fetal brain cDNA library, with greater than 84% homology tothe first homolog. The two ESTs are identical except HGS47815 is 183 bplonger at the 5′ end. HGS47815 contains 3035 bp and the sequencecomparison suggested that it is missing approximately 1 kb of theputative photolyase homolog at the 5′ end. Using this clone as a probe,a hybridization screening was conducted through the human fetal braincDNA library from which HGS6392 and HGS47815 were initially discovered.From this screening, a positive clone (SO5), which was 466 bp longerthan HGS47815, was identified.

[0103] The gene identified previously (Adams et al. (1995); Todo et al.(1996) has been designated hCRY1. In the present specification, thehuman photolyase homolog genes are referred to as hCRY1 and hCRY2 andthe corresponding gene products are referred to at hCRY1 and hCRY2,respectively. These names are in compliance with the nomenclature forblue-light photoreceptors (i.e., cryptochromes) in plants (Short, T. W.et al., Ann. Rev. Plant. Physiol. Plant Mol. Biol. 45:143-171 (1994)).The gene of the present invention has been designated hCRY2.

[0104] The hCRY2 gene was originally identified in a human fetal braincDNA library and was found to be expressed in fibroblasts as well.Further, the hCRY2 gene was detected in human cDNA libraries preparedfrom fetal brain, synovial sarcoma, resting T-cell, infant brain,cerebellum, endometrial tumor, testes tumor, adult retina,chondrosarcoma, breast, and pituitary. Compared to hCRY1, hCRY2 occurredat lower frequency in the human cDNA database in all tissues tested.

[0105] To obtain the entire 5′ terminal part of the hCRY2 gene, the RACEPCR procedure was used. Briefly, a specific primer for the 3 end of thegene, 5′-GGGCTCTGCCACAGGGTGACTGAGGTC-3′ (SEQ ID NO:7), was used forfirst strand cDNA synthesis. First round of PCR amplification for the 5′terminal part of the hCRY2 gene was carried out using a gene specificprimer, 5′-AATACCCGGACCCCGCTC-3′ (SEQ ID NO:8), at the 3′ end of thegene and a degenerative primer at the 5′ end as described by themanufacturer. This was followed by a second round of amplification usinganother gene specific primer, 5′CAGGTCCCACAGGCGGTA-3′ (SEQ ID NO:9) atthe 3′ end of the gene and another degenerative primer at the 5′ end.Sequence comparison of the open reading frame of the amplified productto the first photolyase homolog confirmed that the 5 end of the gene hadbeen cloned.

[0106] A MBP fusion of the S05 clone was constructed by ligating anEcoRI/BglIII fragment containing the entire open reading frame of S05into the EcoRI/HI site of pMal-c2. This construct, which encodes thecarboxy terminal 381 amino acids of the hCRY2 protein (amino acidresidues 191-571 in SEQ ID NO:2), was named pDH1996-2.

[0107] Large scale sequencing of expression sequence tagged (EST) cDNAsrevealed a clone with homology to the microbial photolyase genes. Thisclone was designated a photolyase isolog since there is no convincingevidence that humans have a photolyase which can repair cyclobutanepyrimidine dimers (Adams, M. D. et al., Nature 377:3-174 (1995)).Independently, Todo et al. cloned and sequenced the gene for theapoenzyme of the newly discovered (6-4) photolyase from D. melanogaster(Todo, T. et al., Science 272:109-112 (1996)). It was found that the(6-4) photolyase has high degree of homology with thephotolyase/blue-light photoreceptor family of proteins (Ahmad, M. etal., Nature 366:162-166 (1993); Malhotra,K. et al., Biochemistry34:6892-6899 (1995)), including the human photolyase isolog. In fact,when the entire cDNA of the human photolyase isolog was isolated andsequenced, it revealed an astonishing 48% sequence identity with the D.melanogaster (6-4) photolyase (Todo, T. et al., Science 272:109-112(1996)).

[0108] Sequence comparison of the hCRY1 and hCRY2 proteins, along with arepresentative member of a type I (microbial) class photolyase, theblue-light photoreceptor gene HY4 of A. thaliana, and the (6-4)photolyase of D. melanogaster, are shown in FIGS. 4A-4C. hCRY1 and hCRY2exhibit 65% sequence identity at the nucleotide level and 74% sequenceidentity at the amino acid level. hCRY2 also shows high degree ofsequence homology to D. melanogaster (6-4) photolyase with a 51%sequence identity over the entire length.

[0109] Aside from the high degree of sequence homology between hCRY1 andhCRY2, the most noteworthy feature of the sequences of these proteins isthe complete divergence over the carboxy-terminal 80 amino acids. Asimilar divergence has been found between the two A. thaliana blue-lightphotoreceptors (Ahmad, M. et al., Plant Molec. Biol. 30:851-861 (1996)).It is believed that this “tail” region of the A. thaliana photoreceptorinteracts with an effector molecule (Lin, C. et al., Proc. Natl. Acad.Sci. USA 98:6389-6393 (1995)). It is also believed that the hCRY1 andhCRY2 proteins also interact with downstream targets.

EXAMPLE 2 Purification of Recombinant human CRY Proteins

[0110] hCRY1 and hCRY2 proteins were expressed as MBP fusion proteins,using the MBP fusion vector pMal-c2 (FIG. 5), in DR153 E. coli cells.The hCRY1 construct was called pDH1996-1. The hCRY2 construct was calledpDH1996-2. As discussed supra, pDH1996-2 contains the carboxy terminal381 amino acids (amino acid residues 191-571 in SEQ ID NO:2).Oligonucleotide primers were used to amplify the hCRY2 sequence. The 5′primer sequence was 5′-CGCGAATTCCTCCCTGGAGGAGCTGGG-3′ (SEQ ID NO:10),which contains the underlined EcoR I restriction site followed by 19bases corresponding to nucleotides 635-654 in the sequence set forth inSEQ ID NO:1. The 3′ primer sequence was5′-GCGAGATCTTCAGGCATCCTTGCTCGG-3′ (SEQ ID NO:11), which contains theunderlined Bgl II restriction site, followed by 18 bases reverse andcomplementary to nucleotides 1825-1842 in the sequence set forth in SEQID NO:1.

[0111] A longer MBP-hCRY2 fusion protein, containing amino acid residues−15 to 571 of the sequence set forth in SEQ ID NO:2, was expressed inDR153 cells using the pMal-c2 vector. Oligonucleotide primers were usedto amplify the longer hCRY2 sequence. The 5′ primer sequence was5′-GCGGATATCG-CGGCAGCTGTGGCCCCG-3 (SEQ ID NO: 12), which contains theunderlined EcoR V restriction site followed by 18 bases corresponding tonucleotides 22-39 in the sequence set forth in SEQ ID NO: 1. The 3′primer sequence was 5′-GCGAAGCTTTCAGGCATCCTTGCTCGG-3′ (SEQ ID NO:13),which contains the underlined Hind III restriction site, followed by 18bases reverse and complementary to nucleotides 1825-1842 in the sequenceset forth in SEQ ID NO:1. This amplified fragment was digested with EcoRV and Hind III prior to ligation into the pMal-c2 vector (afterdigestion of the vector with Xmn I and Hind III).

[0112] Expressed proteins were purified by affinity chromatography onamylose resin (Malhotra, K. et al., Biochemistry 34:6892-6899 (1995)).Since the possibility exists that fusion with MBP may interfere withenzymatic function, a MBP fusion form of the D. melanogaster (6-4)photolyase (Todo, T. et al., Science 272: 109-112 (1996)) was used as acontrol. As discussed supra, the (6-4) photolyase is highly homologousto the hCRY1 and hCRY2 photoreceptors and was prepared and purified aswere the hCRY1 and hCRY2 proteins.

EXAMPLE 3 Spectroscopic Properties of hCRY1 and hCRY2

[0113] All photolyases and blue-light photoreceptors that have beencharacterized to date contain FAD and a second chromophore, which is afolate in most organisms. In a few species which can synthesizedeazaflavin, the second photolyase chromophore is deazariboflavin (Eker,A. P. et al., J. Biol. Chem. 265:8009-8015 (1990); Malhotra, K. et al.,Biochemistry 34: 6892-6899 (1995)). hCRY1 and hCRY2 were assayed for thepresence of chromophores. The absorption spectra of the purified hCRY1and hCRY2 proteins were recorded with a Hewlett-Packard Model 8451Aspectrophotometer and the fluorescence spectra of the chromophores weremeasured at 22° C. in a Shimadzu RF5000 U Spectrofluorometer.

[0114] The absorption spectra of the MBP fusion forms of hCRY 1 andhCRY2 (amino acids residues 191-571 in SEQ ID NO:2) are shown in FIG.6A. Both proteins exhibited a distinct 420 nm peak with residualabsorption extending all the way to 700 nm. The absorption spectra werealmost identical to the absorption spectra of the cyclobutane pyrimidinedimer photolyase (Kim, S. T. et al., Mutation Res. 363:97-104 (1996))and the (6-4) photolyase (data not shown) from D. melanogaster.

[0115] It has been demonstrated that the D. melanogaster T<>T photolyasecontained FAD and folate as chromophores (Kim, S. T. et al., MutationRes. 363:97-104 (1996)). Hence, it was reasoned that hCRY1 and hCRY2 mayalso contain these cofactors. A simple assay revealed that this isindeed the case. hCRY1 and hCRY2 were denatured by heating for 10minutes at 65° C. in 0.1 M HCL and 0.8% SDS. Following centrifugation toremove the protein precipitate, excitation and emission fluorescencespectra were recorded. FIG. 6B shows a diagnostic flavin fluorescencespectrum. It was concluded that both hCRY1 and hCRY2 contain flavin.Furthermore, upon increasing the pH to 10 by addition of NaOH, theflavin fluorescence was severely quenched, further confirming thecofactor as FAD (Faeder, E. J., Anal. Biochem. 53:332-336 (1973)).

[0116] Alkaline pH had another notable effect on the fluorescencespectrum: a new species with an excitation maximum at 380 and emissionmaximum at 470 appeared (FIG. 6C). This behavior is typical of reducedpterin, which is non-fluorescent but is converted to highly fluorescentoxidized pterin upon incubation in alkaline solutions (Johnson, J. L. etal., Proc. Natl. Acad. Sci. USA 85:2046-2050 (1988)). Furthermore, theexcitation and emission spectra of the second chromophore are identicalto that of the D. melanogaster T<>T photolyase, which was shown to be afolate by TLC analysis with appropriate standards (Kim, S. T. et al.,Mutation Res. 363:97-104 (1996)). Thus, it was concluded that hCRY1 andhCRY2, like other members of the photolyase/photoreceptor family,contain FAD and a pterin as the two chromophore/cofactors.

EXAMPLE 4 Photolyase Activity Assay

[0117] In a photolyase assay, the restoration of the susceptibility tocleavage of the TTAA sequence by the MseI restriction endonuclease wasmeasured in a DNA fragment where the TT is either in the form of acyclobutane thymine dimer (T<>T) or (6-4) photoproduct (Malhotra, K. etal., Biochemistry 34:6892-6899 (1995); Kim, S. T. et al., Photochem.Photobiol. 63:292-295 (1996)). A 54 mer oligonucleotide duplex, and a49mer oligonucleotide duplex, containing a centrally located T<>T andT[6-4]T, respectively, were prepared as described previously (Smith, C.A., J. Biol. Chem. 268:11143-11151 (1993)) and were kindly provided byDr. J. S. Taylor (Washington University).

[0118] In the photoreactivation assay, hCRY1 and hCRY 2 (amino acidsresidues 191-571 in SEQ ID NO:2) proteins (40 nM) were mixed with 0.5 nMsubstrate in a 50 μl reaction containing 50 mM Tris pH 7.4, 100 mM NaCl,6 mM dithiothreitol, 2 mM EDTA, 5 μg bovine serum albumin and 5%glycerol. The mixture was incubated in the dark at room temperature for10 minutes and then exposed to photoreactivating light (λ_(max)=366 nm),at 4° C. for 1 hour, from a Sylvania black light (Model B-100) at afluence rate of 2 milliwatts/cm2. The DNA was then extracted withphenol/chloroform, precipitated with ethanol, resuspended in restrictionenzyme buffer and digested with 8 units of MseI for 1 hour. The reactionproducts were electrophoresed on a 8% denaturing gel and the level ofdigested (repaired) DNA was determined by a Phospholmager (MolecularDynamics Inc.). For the (6-4) photoproduct, the level of 21mer detectedindicated the extent of repair. For the T<>T substrate, the level of19mer detected indicated the extent of repair.

[0119] The spectroscopic properties of hCRY1 and hCRY2 (supra) wereconsistent with these proteins being a Pyr<>Pyr photolyase, a (6-4)photolyase, or a photoreceptor. To differentiate between thesepossibilities, the recombinant proteins were tested for repair activity.E. coli photolyase repaired a T<>T photoproduct. 54% of the T< >Tsubstrate was repaired. However, both CRY1 and hCRY2 failed to repairthe T<>T photoproduct.

[0120]D. melanogaster (6-4) photolyase repaired a T[6-4]T photoproduct.48% of the photoproduct was repaired. In contrast, both CRY1 and hCRY2failed to repair the T[6-4]T photoproduct. After conducting the repairexperiments were under a variety of conditions (higher proteinconcentration and higher dose of photoreactivating light), it wasconcluded that hCRY1 and hCRY2 cannot have more than 0.1% of thephotolyase activities detected with bona fide photolyases. Thus, it wasconcluded that the recombinant photolyase homologs do not havephotolyase activity.

[0121] Even though these data strongly suggest that hCRY1 and hCRY2 arenot photolyases, it is conceivable that the proteins expressed inheterologous system were somewhat misfolded or lacked aposttranslational modification necessary for activity. Hence, thenatural sources were tested for activity. Cell-free extracts fromfibroblasts (T093), which expressed hCRY1 and hCRY2 (as revealed byprimer extension (for both hCRY1 and hCRY2) and immunoblotting (forhCRY1 only), failed to show any (6-4) photolyase activity.

[0122] To ascertain whether this lack of activity was due to inhibitionby other proteins known to exist in cell-free extracts which bind to(6-4) photoproduct (Chu, G. et al., Science 242:564-567 (1988); Ghosh,R. et al., Proc. Natl. Acad. Sci. USA 93:6918-6923 (1996); Wakasugi, M.et al., Nucl. Acids Res. 24:1099-1104 (1996)), Drosophila (6-4)photolyase was mixed with the fibroblast cell free extract and thismixture was assayed for photoreactivation activity using a T(6-4)Tsubstrate. Assays with human cell free extract (infra) were performed ina similar manner as describe supra, except that 50 μg of CFE was used inthe reaction.

[0123] In the absence of cell-free extract, Drosophila (6-4) photolyaserepaired 38% of the T(6-4) substrate. In the presence of cell-freeextract, Drosophila (6-4) photolyase repaired 29% of the substrate. Thislevel of inhibition cannot explain the total lack of photolyase activityin the cell-free extract, which was assayed under a variety ofconditions.

[0124] Finally, hCRY1 purified from a baculovirus/insect cell expressionsystem also failed to show any photolyase activity (data not shown).Thus, hCRY1 and hCRY2 do not appear to possess a photolyase activity.

[0125] The longer form of the MBP-hCRY2 fusion protein, containing aminoacids −15 to 571 in SEQ ID NO:2, displayed spectral properties similarto those of the MBP-hCRY2 fusion protein that contained only amino acidresidues 191-571. Like the shorter MBP-hCRY2 fusion protein, the longerMBP-hCRY2 fusion protein failed to exhibit photolyase activity.

[0126] Many attempts from several labs to detect and isolate photolyasesfrom human cells have failed, leading to a near-consensus in the fieldthat humans do not have photolyase (Ley, R. D., Proc. Natl. Acad. Sci.USA 98:4337 (1993); Li, Y. F. et al., Proc. Natl. Acad. Sci. USA90:4389-4393 (1993); Kato, T. et al., Nucl. Acids. Res. 22:4119-4124(1994)). However, the recent discovery of a photolyase for (6-4)photoproducts, and the finding that it belongs in thephotolyase/photoreceptor family of proteins (Todo, T. et al., Nature361:371-374 (1996)), raised the interesting possibility that humansmight have a (6-4) photolyase. Furthermore, a human homolog, with 48%sequence identity with D. melanogaster (6-4) photolyase, was identified(Todo et al., (1996)). The encoded protein could have been the elusivecyclobutane pyrimidine dimer photolyase described by Sutherland et al.,(Proc. Natl. Acad. Sci. USA 92:9732-9736 (1996)), a (6-4) photolyasewhich had not been searched for in humans in a systematic way, or aphotoreceptor. This present work was undertaken to differentiate betweenthese possibilities.

[0127] The results of the present work clearly show that the human (6-4)photolyase homolog identified previously (hCRY1) and the new homologidentified herein (hCRY2) are neither (6-4) photolyase nor cyclobutanepyrimidine dimer photolyase. Work with recombinant proteins and humancell free extract showed that the proteins encoded by these two genesneither bind to UV damaged DNA (data not shown) nor repair T<>T orT[6-4]T photoproducts in the absence or presence of light. Thus, one isleft with the third alternative, that the proteins encoded by the hCRY1and hCRY2 genes function as blue-light photoreceptors.

EXAMPLE 5 Cloning and Expression of hCRY2 Protein in a BaculovirusExpression System

[0128] In this illustrative example, the plasmid shuttle vector pA2 isused to insert the cloned DNA encoding the complete protein, includingits naturally associated secretary signal (leader) sequence, into abaculovirus to express the mature hCRY2 protein, using standard methodsas described in Summers et al., A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperimental Station Bulletin No. 1555 (1987). This expression vectorcontains the strong polyhedrin promoter of the Autographa californicanuclear polyhedrosis virus (AcMNPV) followed by convenient restrictionsites such as BamH I and Asp 718. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak promoter inthe same orientation, followed by the polyadenylation signal of thepolyhedrin gene. The inserted genes are flanked on both sides by viralsequences for cell-mediated homologous recombination with wild-typeviral DNA to generate viable virus that express the clonedpolynucleotide.

[0129] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

[0130] The cDNA sequence encoding the full length hCRY2 protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in SEQ ID NO:2, is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3 sequences of thegene. The 5′ primer has the sequence5′-GCGAGATCTCCGCCATCATGGCGGCAACTGTGGCAAC-3′ (SEQ ID NO: 14) containingthe underlined Bgl II restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 20 bases of thesequence of the complete hCRY2 protein shown in SEQ ID NO:1, beginningwith the AUG initiation codon. The 3′ primer has the sequence5′-GCGTCTAGATCAGGCATCCTTGCTCGG-3′ (SEQ ID NO: 15) containing theunderlined Xba I restriction site, followed by 18 nucleotides reverseand complementary to nucleotides 1825-1842 in SEQ ID NO:1

[0131] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Bgl II and Xba I and againis purified on a 1% agarose gel. This fragment is designated herein“F1”.

[0132] The pA2 vector is digested with the restriction enzymes BamH Iand Xba I and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “VI”.

[0133] Fragment F1 and the dephosphorylated plasmid VI are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the humanhCRY2 gene using the PCR method, in which one of the primers that isused to amplify the gene and the second primer is from well within thevector so that only those bacterial colonies containing hCRY2 genefragment will show amplification of the DNA. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBachCRY2.

[0134] Five μg of the plasmid pBachCRY2 is co-transfected with 1.0 μg ofa commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBachCRY2 are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours, the transfection solution is removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. The plate is put back into an incubator and cultivationis continued at 27° C. for four days.

[0135] After four days, the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later, the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-hCRY2.

[0136] To verify the expression of the hCRY2 gene, Sf9 cells are grownin Grace's medium supplemented with 10% heat inactivated FBS. The cellsare infected with the recombinant baculovirus V-hCRY2 at a multiplicityof infection (“MOI”) of about 2. Six hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). If radiolabeledproteins are desired, 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then they are harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinsare analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature protein and thus the cleavage point and length of thesecretory signal peptide.

EXAMPLE 6 Cloning and Expression of hCRY2 in Mammalian Cells

[0137] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0138] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells.

[0139] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem. J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) and NSO cells are often used forthe production of proteins.

[0140] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

EXAMPLE 6(a) Cloning and Expression in COS Cells

[0141] The expression plasmid, phCRY2 HA, is made by cloning a cDNAencoding hCRY2 into the expression vector pcDNAI/Amp or pcDNAIII (whichcan be obtained from Invitrogen, Inc.).

[0142] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0143] A DNA fragment encoding the hCRY2 is cloned into the polylinkerregion of the vector so that recombinant protein expression is directedby the CMV promoter. The plasmid construction strategy is as follows.The hCRY2 cDNA of the deposited clone is amplified using primers thatcontain convenient restriction sites, much as described above forconstruction of vectors for expression of hCRY2 in E. coli. Suitableprimers include the following, which are used in this example. The 5′primer, containing the underlined Bgl II site, a Kozak sequence, an AUGstart codon and 20 bases of the 5′ coding region of the hCRY2 has thefollowing sequence: 5′-GCGAGATCTCCGCCATCATGGCG-GCAACTGTGGCAAC-3′ (SEQ IDNO:14). The 3′ primer, containing the underlined Xho I site, followed by18 bp reverse and complementary to nucleotides 1822-1842 of thenucleotide sequence set forth in SEQ ID NO:1 has the following sequence:5′-GCGCTCGAGTCAGGCATCCTTGCTCGGCCAG-3′ (SEQ ID NO:16).

[0144] The PCR amplified DNA fragment is digested with Bgl II and Xho I.The vector, pcDNA3/Amp, is digested with BamH I and Xho I. The PCRamplified DNA fragment and the linearized vector are then ligated. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037), and the transformed culture is plated on ampicillin mediaplates which then are incubated to allow growth of ampicillin resistantcolonies. Plasmid DNA is isolated from resistant colonies and examinedby restriction analysis or other means for the presence of thehCRY2-encoding fragment.

[0145] For expression of recombinant hCRY2, COS cells are transfectedwith an expression vector, as described above, using DEAE-Dextran, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression ofhCRY2 by the vector.

[0146] Expression of the hCRY2-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing 35S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

EXAMPLE 6(b) Cloning and Expression in CHO Cells

[0147] The vector pC4 is used for the expression of hCRY2 protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., J Biol. Chem.253:1357-1370 (1978), Hamlin, J. L. and Ma, C., Biochem. et Biophys.Acta, 1097:107-143 (1990), Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

[0148] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter are Bam HI, XbaI,and Asp718 restriction enzyme cleavage sites that allow integration ofthe genes. Behind these cloning sites the plasmid contains the 3′ intronand polyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express hCRY2 in a regulated way in mammaliancells (Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551(1992)). For the polyadenylation of the mRNA other signals,e.g., from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0149] The plasmid pC4 is digested with the restriction enzymes BamH Iand Xba I and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

[0150] The DNA sequence encoding the complete hCRY2 protein includingits leader sequence is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene. The 5′ primer hasthe sequence 5′-GCGAGATCTCCGCCATCATGGCGGCAACTGTGGCAAC-3′ (SEQ ID NO:14)containing the underlined Bgl II restriction enzyme site followed by anefficient signal for initiation of translation in eukaryotes, asdescribed by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 22 basescorresponding to nucleotides −2 to 20 of SEQ ID NO:1. The 3′ primer hasthe sequence 5′-GCGTCTAGATCAGGCATCCTTGCTCGG-3′ (SEQ ID NO:15),containing the underlined Xba I restriction site followed by a stopcodon and 18 nucleotides reverse and complementary to nucleotide1825-1842 in the sequence set forth in SEQ ID NO:1.

[0151] The amplified fragment is digested with the endonucleases Bgl IIand Xba I and is then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli H13101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0152] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days, single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

EXAMPLE 7 Tissue Distribution of hCRY2 mRNA Expression

[0153] Northern blot analysis was carried out to examine hCRY2 geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the hCRY2 protein (SEQ ID NO:1) was labeled with³²p using the Rediprime™ DNA labeling system (Amersham Life Science,Arlington, Ill.), according to manufacturer's instructions. Afterlabeling, the probe was purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT1200-1. The purified labeled probe was then used to examinevarious human tissues for hCRY2 mRNA.

[0154] Multiple Tissue Northern (MTN) blots containing various humantissues were obtained from Clontech and were examined with the labeledprobe using ExpressHyb™ hybridization solution (Clontech) according tomanufacturer's protocol number PT1190-1. Following hybridization andwashing, the blots were mounted and exposed to film at −70° C.overnight, and films developed according to standard procedures.

[0155] Multiple, stronger signals, ranging size from about 1.4 togreater than about 9.5 kB, were detected in lanes corresponding to RNAfrom heart, brain, skeletal muscle, and pancreas. Weaker signals,ranging in size from about 1.4 to about 4 kb were detected in lanescorresponding to lung and kidney.

[0156] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0157] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0158] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 22 1 4185 DNA Homo sapiens CDS (51)..(1829) sig_peptide (51)..(116)mat_peptide (117)..(1829) 1 ggccacgcgt cgactagtac gggggggggg ggggggcattctggacagtc atg gcg 56 Met Ala gca act gtg gca acg gcg gca gct gtg gccccg gcg cca gcg ccc ggc 104 Ala Thr Val Ala Thr Ala Ala Ala Val Ala ProAla Pro Ala Pro Gly -20 -15 -10 -5 acg gac agc gcc tct tcg gtg cac tggttc cgc aaa ggg ctg cga ctc 152 Thr Asp Ser Ala Ser Ser Val His Trp PheArg Lys Gly Leu Arg Leu -1 1 5 10 cac gac aac ccg gcg ttg ctg gcg gccgtg cgc ggg gcg cgc tgc gtg 200 His Asp Asn Pro Ala Leu Leu Ala Ala ValArg Gly Ala Arg Cys Val 15 20 25 cgc tgc gtt tac att ctc gac ccg tgg ttcgcg gcc tcc tcc tca gtc 248 Arg Cys Val Tyr Ile Leu Asp Pro Trp Phe AlaAla Ser Ser Ser Val 30 35 40 ggg atc aac cga tgg agg ttc cta ctt cag tctctg gaa gat ttg gac 296 Gly Ile Asn Arg Trp Arg Phe Leu Leu Gln Ser LeuGlu Asp Leu Asp 45 50 55 60 aca agt tta agg aaa ctg aac tcc cgc ctg tttgta gtc cgg gga cag 344 Thr Ser Leu Arg Lys Leu Asn Ser Arg Leu Phe ValVal Arg Gly Gln 65 70 75 cca gcc gac gtg ttc cca agg ctg ttc aag gaa tgggga gtg acc cgc 392 Pro Ala Asp Val Phe Pro Arg Leu Phe Lys Glu Trp GlyVal Thr Arg 80 85 90 ttg acc ttt gaa cat gac tct gaa ccc ttt ggg aaa gaacgg gat gca 440 Leu Thr Phe Glu His Asp Ser Glu Pro Phe Gly Lys Glu ArgAsp Ala 95 100 105 gcc atc atg aag atg acc aag gag gct ggt gtg gaa gtagtg acg gag 488 Ala Ile Met Lys Met Thr Lys Glu Ala Gly Val Glu Val ValThr Glu 110 115 120 aat tct cat acc ctc tat gac ctg gac agg atc att gagctg aat ggg 536 Asn Ser His Thr Leu Tyr Asp Leu Asp Arg Ile Ile Glu LeuAsn Gly 125 130 135 140 cag aag cca ccc ctt aca tac aag cgc ttt cag gccatc atc agc cgc 584 Gln Lys Pro Pro Leu Thr Tyr Lys Arg Phe Gln Ala IleIle Ser Arg 145 150 155 atg gag ctg ccc aag aag cca gtg ggc ttg gtg accagc cgg cag atg 632 Met Glu Leu Pro Lys Lys Pro Val Gly Leu Val Thr SerArg Gln Met 160 165 170 gag agc tgc agg gcc gag atc cag gag aac cac gacgag acc tac ggc 680 Glu Ser Cys Arg Ala Glu Ile Gln Glu Asn His Asp GluThr Tyr Gly 175 180 185 gtg ccc tcc ctg gag gag ctg ggg ttc ccc act gaagga ctt ggt cca 728 Val Pro Ser Leu Glu Glu Leu Gly Phe Pro Thr Glu GlyLeu Gly Pro 190 195 200 gct gtc tgg cag gga gga gag aca gaa gct ctg gcccgc ctg gat aag 776 Ala Val Trp Gln Gly Gly Glu Thr Glu Ala Leu Ala ArgLeu Asp Lys 205 210 215 220 cac ttg gaa cgg aag gcc tgg gtt gcc aac tatgag aga ccc cga atg 824 His Leu Glu Arg Lys Ala Trp Val Ala Asn Tyr GluArg Pro Arg Met 225 230 235 aac gcc aac tcc ctc ctg gcc agc ccc aca ggcctc agc ccc tac ctg 872 Asn Ala Asn Ser Leu Leu Ala Ser Pro Thr Gly LeuSer Pro Tyr Leu 240 245 250 cgc ttt ggt tgt ctc tcc tgc cgc ctc ttc tactac cgc ctg tgg gac 920 Arg Phe Gly Cys Leu Ser Cys Arg Leu Phe Tyr TyrArg Leu Trp Asp 255 260 265 ctg tat aaa aag gtg aag cgg aac agc aca cctccc ctc tcc cta ttt 968 Leu Tyr Lys Lys Val Lys Arg Asn Ser Thr Pro ProLeu Ser Leu Phe 270 275 280 ggg caa ctc cta tgg cga gag ttc ttc tac acggca gct acc aac aac 1016 Gly Gln Leu Leu Trp Arg Glu Phe Phe Tyr Thr AlaAla Thr Asn Asn 285 290 295 300 ccc agg ttt gac cgc atg gag ggg aac cccatc tgc atc cag atc ccc 1064 Pro Arg Phe Asp Arg Met Glu Gly Asn Pro IleCys Ile Gln Ile Pro 305 310 315 tgg gac cgc aat cct gag gcc ctg gcc aagtgg gct gag ggc aag aca 1112 Trp Asp Arg Asn Pro Glu Ala Leu Ala Lys TrpAla Glu Gly Lys Thr 320 325 330 ggc ttc cct tgg att gat gcc atc atg acccaa ctg agg cag gag ggc 1160 Gly Phe Pro Trp Ile Asp Ala Ile Met Thr GlnLeu Arg Gln Glu Gly 335 340 345 tgg atc cac cac ctg gcc cgg cat gcc gtggcc tgc ttc ctg acc cgc 1208 Trp Ile His His Leu Ala Arg His Ala Val AlaCys Phe Leu Thr Arg 350 355 360 ggg gac ctc tgg gtc agc tgg gag agc ggggtc cgg gta ttt gat gag 1256 Gly Asp Leu Trp Val Ser Trp Glu Ser Gly ValArg Val Phe Asp Glu 365 370 375 380 ctg ctc ctg gat gca gat ttc agc gtgaac gca ggc agc tgg atg tgg 1304 Leu Leu Leu Asp Ala Asp Phe Ser Val AsnAla Gly Ser Trp Met Trp 385 390 395 ctg tcc tgc agt gct ttc ttc cag cagttc ttc cac tgc tac tgc cct 1352 Leu Ser Cys Ser Ala Phe Phe Gln Gln PhePhe His Cys Tyr Cys Pro 400 405 410 gtg ggc ttt ggc cgt cgc acg gac cccagt ggg gac tac atc agg cga 1400 Val Gly Phe Gly Arg Arg Thr Asp Pro SerGly Asp Tyr Ile Arg Arg 415 420 425 tac ctg ccc aaa ttg aaa gcg ttc ccctct cga tac atc tat gag ccc 1448 Tyr Leu Pro Lys Leu Lys Ala Phe Pro SerArg Tyr Ile Tyr Glu Pro 430 435 440 tgg aat gcc cca gag tca att cag aaggca gcc aag tgc atc att ggt 1496 Trp Asn Ala Pro Glu Ser Ile Gln Lys AlaAla Lys Cys Ile Ile Gly 445 450 455 460 gtg gac tac cca cgg ccc atc gtcaac cat gcc gag acc agc cgg ctt 1544 Val Asp Tyr Pro Arg Pro Ile Val AsnHis Ala Glu Thr Ser Arg Leu 465 470 475 aac att gaa cga atg aag cag atttac cag cag ctt tcg cgc tac cgg 1592 Asn Ile Glu Arg Met Lys Gln Ile TyrGln Gln Leu Ser Arg Tyr Arg 480 485 490 gga ctc tgt cta ctg gca tct gtccct tcc tgt gtg gaa gac ctc agt 1640 Gly Leu Cys Leu Leu Ala Ser Val ProSer Cys Val Glu Asp Leu Ser 495 500 505 cac cct gtg gca gag ccc agc tcgagc cag gct ggc agc atg agc agt 1688 His Pro Val Ala Glu Pro Ser Ser SerGln Ala Gly Ser Met Ser Ser 510 515 520 gca ggc cca aga cca cta ccc agtggc cca gca tcc ccc aaa cgc aag 1736 Ala Gly Pro Arg Pro Leu Pro Ser GlyPro Ala Ser Pro Lys Arg Lys 525 530 535 540 ctg gaa gca gcc gag gaa ccacct ggt gaa gaa ctc agc aaa cgg gcc 1784 Leu Glu Ala Ala Glu Glu Pro ProGly Glu Glu Leu Ser Lys Arg Ala 545 550 555 cgg gtg gca gag ttg cca acccca gag ctg ccg agc aag gat gcc 1829 Arg Val Ala Glu Leu Pro Thr Pro GluLeu Pro Ser Lys Asp Ala 560 565 570 tgagactgca gagcccttgc tccgtgagcaaagcctgggt gcccaagcag ccaccgcagc 1889 agcagagtac aacctgcaga gaagctgatcaccgggcaga gatagagcga gcatgtgtgt 1949 gtgtgtgcgc gtgtgcagag gagggagtggtgtgcctgtt tgtgtgtgca tgcatctgtt 2009 gacactcatg attctgaatg ttgcctgggctgggggagta cctgtagcac gccagtgctg 2069 tttcccggcc tccagacaca aggctcgaggttatggcagt gactttcagc tgagacctgt 2129 tcctgcaagc cagctgcctt gtctgaacagaacgtagtgg taggacccta gctgggattc 2189 tggcatctgc ctccctagac ctccttccctccctcctcac gtcaggctgt ggagcaggag 2249 cacagcagtt ctggctgttg tccaaagcatgggattctgg aggcagccag agccctgctg 2309 agttcctgct ttctgacctg gaggctgagcaggccggagt ggatggatgc tgtccagacg 2369 tagccacctg gcctctgttt cttattttaaaattctctgc tactgggctc agtcccaggc 2429 ccttccttgg gcttctggga ctgagcatgaggccatagac agatctaaaa agtttccacc 2489 accctacaga agtacacaca gatacctgactggtgtgggg tatgcctggt actgtaatag 2549 gagcctaaga cagcacacct accttttcaggatttagaac ctaaaattag aaagagaatc 2609 ccagctgtca ttgttccttc cccagaagctaagagccagc ctcagagcct acccaggagc 2669 tgtgaagggg caagggtcaa actgactcactctaccagga ggagaccagg ttgcagtggc 2729 gtaaggcccc ctggtttctc tggccacactccaaggcacc acagtgctgc cagtgaggac 2789 agctgacacc cagccaggga aaccattctagtctttattc tgttggcttc cagggcctgt 2849 cctgaacttg tcagcatcca gactgccatgtcagctatcc cagtagctga gctccaagga 2909 ctcaggcaga gggactcagg gatggggactgccaggggca gttggcaaaa gtccaagtag 2969 agattacacc cagaacacca ttccttccaggagcagtagg tgggaggttt gacccagaga 3029 agccaatcct tgcattccag gagtggcctgtgcctcccac ctcttccttc ccactgccaa 3089 aggcctgtgt tgagaaagat gtcatgcaaaaggacgacgg tggccaacta aagcaagtct 3149 tcctaccacc ctgtggcctg cacttgagccacaaagtgtg tgtgtgtgtg tgcgtgtgtg 3209 gtaagtgtgt gtgtgtgtgg ctatgaggctgattcctgtt tggatttttg tcctcacgtg 3269 tatcattaag ctggcctttg ggccttttcctttctacctc ccctgtgacc tttcctagcc 3329 tcagatctgt taattctttt ggccccagccctgtccctca ctgtcctctg tccttggacc 3389 agaaccctgg ggtcagaccc atctcctgtagctgtccatc acactgacag gcttcttcct 3449 gagatatcct caggttttct cagccagagagctgccttta gagtccaact gttgtacgta 3509 tgtcaccttc actagaaatg tcccatcatcgtgggagggg agcagggcac aggggatggt 3569 gtgcattcag agcattgggt tgggggcttccctgttccct cagccccagt cgagaggaaa 3629 gagaatcggg ccactgccag aaagagagtcaagcaaacct ggaagggcaa atctgagagt 3689 gggaaggcca aaggccgagg cccagatttagtattcacta gcagcgcctt cgggtagcag 3749 gatgattcct tttcctgcct gtctgctgctggctctcttc cctaaggtac aggttggcag 3809 gaccacctcc gcctacttct ccaccatccctagcatgcca gcccgttccc agatcaacct 3869 gccagtggag tcaggcagtg cactcctggagccaagaggg aagggcaggg tagagagggt 3929 atgtccagta gcctggagct ccatggtggcttcatgcctc ccttctccca gctcaggtgg 3989 ccctgagggc tccctcggaa cagtgcctcaaatcctgacc caagggccag catggggaag 4049 agatggttgc aggcaaaatg cactttatagagattttcta ttgctgggaa ggtgtgtttc 4109 tcccacaatt tgtttgtgaa tattcacttgttttataaat gtctgacctg tcttgagtaa 4169 aaaaaaaaaa aaaaaa 4185 2 593 PRTHomo sapiens 2 Met Ala Ala Thr Val Ala Thr Ala Ala Ala Val Ala Pro AlaPro Ala -20 -15 -10 Pro Gly Thr Asp Ser Ala Ser Ser Val His Trp Phe ArgLys Gly Leu -5 -1 1 5 10 Arg Leu His Asp Asn Pro Ala Leu Leu Ala Ala ValArg Gly Ala Arg 15 20 25 Cys Val Arg Cys Val Tyr Ile Leu Asp Pro Trp PheAla Ala Ser Ser 30 35 40 Ser Val Gly Ile Asn Arg Trp Arg Phe Leu Leu GlnSer Leu Glu Asp 45 50 55 Leu Asp Thr Ser Leu Arg Lys Leu Asn Ser Arg LeuPhe Val Val Arg 60 65 70 Gly Gln Pro Ala Asp Val Phe Pro Arg Leu Phe LysGlu Trp Gly Val 75 80 85 90 Thr Arg Leu Thr Phe Glu His Asp Ser Glu ProPhe Gly Lys Glu Arg 95 100 105 Asp Ala Ala Ile Met Lys Met Thr Lys GluAla Gly Val Glu Val Val 110 115 120 Thr Glu Asn Ser His Thr Leu Tyr AspLeu Asp Arg Ile Ile Glu Leu 125 130 135 Asn Gly Gln Lys Pro Pro Leu ThrTyr Lys Arg Phe Gln Ala Ile Ile 140 145 150 Ser Arg Met Glu Leu Pro LysLys Pro Val Gly Leu Val Thr Ser Arg 155 160 165 170 Gln Met Glu Ser CysArg Ala Glu Ile Gln Glu Asn His Asp Glu Thr 175 180 185 Tyr Gly Val ProSer Leu Glu Glu Leu Gly Phe Pro Thr Glu Gly Leu 190 195 200 Gly Pro AlaVal Trp Gln Gly Gly Glu Thr Glu Ala Leu Ala Arg Leu 205 210 215 Asp LysHis Leu Glu Arg Lys Ala Trp Val Ala Asn Tyr Glu Arg Pro 220 225 230 ArgMet Asn Ala Asn Ser Leu Leu Ala Ser Pro Thr Gly Leu Ser Pro 235 240 245250 Tyr Leu Arg Phe Gly Cys Leu Ser Cys Arg Leu Phe Tyr Tyr Arg Leu 255260 265 Trp Asp Leu Tyr Lys Lys Val Lys Arg Asn Ser Thr Pro Pro Leu Ser270 275 280 Leu Phe Gly Gln Leu Leu Trp Arg Glu Phe Phe Tyr Thr Ala AlaThr 285 290 295 Asn Asn Pro Arg Phe Asp Arg Met Glu Gly Asn Pro Ile CysIle Gln 300 305 310 Ile Pro Trp Asp Arg Asn Pro Glu Ala Leu Ala Lys TrpAla Glu Gly 315 320 325 330 Lys Thr Gly Phe Pro Trp Ile Asp Ala Ile MetThr Gln Leu Arg Gln 335 340 345 Glu Gly Trp Ile His His Leu Ala Arg HisAla Val Ala Cys Phe Leu 350 355 360 Thr Arg Gly Asp Leu Trp Val Ser TrpGlu Ser Gly Val Arg Val Phe 365 370 375 Asp Glu Leu Leu Leu Asp Ala AspPhe Ser Val Asn Ala Gly Ser Trp 380 385 390 Met Trp Leu Ser Cys Ser AlaPhe Phe Gln Gln Phe Phe His Cys Tyr 395 400 405 410 Cys Pro Val Gly PheGly Arg Arg Thr Asp Pro Ser Gly Asp Tyr Ile 415 420 425 Arg Arg Tyr LeuPro Lys Leu Lys Ala Phe Pro Ser Arg Tyr Ile Tyr 430 435 440 Glu Pro TrpAsn Ala Pro Glu Ser Ile Gln Lys Ala Ala Lys Cys Ile 445 450 455 Ile GlyVal Asp Tyr Pro Arg Pro Ile Val Asn His Ala Glu Thr Ser 460 465 470 ArgLeu Asn Ile Glu Arg Met Lys Gln Ile Tyr Gln Gln Leu Ser Arg 475 480 485490 Tyr Arg Gly Leu Cys Leu Leu Ala Ser Val Pro Ser Cys Val Glu Asp 495500 505 Leu Ser His Pro Val Ala Glu Pro Ser Ser Ser Gln Ala Gly Ser Met510 515 520 Ser Ser Ala Gly Pro Arg Pro Leu Pro Ser Gly Pro Ala Ser ProLys 525 530 535 Arg Lys Leu Glu Ala Ala Glu Glu Pro Pro Gly Glu Glu LeuSer Lys 540 545 550 Arg Ala Arg Val Ala Glu Leu Pro Thr Pro Glu Leu ProSer Lys Asp 555 560 565 570 Ala 3 586 PRT Homo sapiens 3 Met Gly Val AsnAla Val His Trp Phe Arg Lys Gly Leu Arg Leu His 1 5 10 15 Asp Asn ProAla Leu Lys Glu Cys Ile Gln Gly Ala Asp Thr Ile Arg 20 25 30 Cys Val TyrIle Leu Asp Pro Trp Phe Ala Gly Ser Ser Asn Val Gly 35 40 45 Ile Asn ArgTrp Arg Phe Leu Leu Gln Cys Leu Glu Asp Leu Asp Ala 50 55 60 Asn Leu ArgLys Leu Asn Ser Arg Leu Phe Val Ile Arg Gly Gln Pro 65 70 75 80 Ala AspVal Phe Pro Arg Leu Phe Lys Glu Trp Asn Ile Thr Lys Leu 85 90 95 Ser IleGlu Tyr Asp Ser Glu Pro Phe Gly Lys Glu Arg Asp Ala Ala 100 105 110 IleLys Lys Leu Ala Thr Glu Ala Gly Val Glu Val Ile Val Arg Ile 115 120 125Ser His Thr Leu Tyr Asp Leu Asp Lys Ile Ile Glu Leu Asn Gly Gly 130 135140 Gln Pro Pro Leu Thr Tyr Lys Arg Phe Gln Thr Leu Ile Ser Lys Met 145150 155 160 Glu Pro Leu Glu Ile Pro Val Glu Thr Ile Thr Ser Glu Val IleGlu 165 170 175 Lys Cys Thr Thr Pro Leu Ser Asp Asp His Asp Glu Lys TyrGly Val 180 185 190 Pro Ser Leu Glu Glu Leu Gly Phe Asp Thr Asp Gly LeuSer Ser Ala 195 200 205 Val Trp Pro Gly Gly Glu Thr Glu Ala Leu Thr ArgLeu Glu Arg His 210 215 220 Leu Glu Arg Lys Ala Trp Val Ala Asn Phe GluArg Pro Arg Met Asn 225 230 235 240 Ala Asn Ser Leu Leu Ala Ser Pro ThrGly Leu Ser Pro Tyr Ile Arg 245 250 255 Phe Gly Cys Leu Ser Cys Arg LeuPhe Tyr Phe Lys Leu Thr Asp Leu 260 265 270 Tyr Lys Lys Val Lys Lys AsnSer Ser Pro Pro Leu Ser Leu Tyr Gly 275 280 285 Gln Leu Leu Trp Arg GluPhe Phe Tyr Thr Ala Ala Thr Asn Asn Pro 290 295 300 Arg Phe Asp Lys MetGlu Gly Asn Pro Ile Cys Val Gln Ile Pro Trp 305 310 315 320 Asp Lys AsnPro Glu Ala Leu Ala Lys Trp Ala Glu Gly Arg Thr Gly 325 330 335 Phe ProTrp Ile Asp Ala Ile Met Thr Gln Leu Arg Gln Glu Gly Trp 340 345 350 IleHis His Leu Ala Arg His Ala Val Ala Cys Phe Leu Thr Arg Gly 355 360 365Asp Leu Trp Ile Ser Trp Glu Glu Gly Met Lys Val Phe Glu Glu Leu 370 375380 Ile Leu Asp Ala Asp Trp Ser Ile Asn Ala Gly Ser Trp Met Trp Leu 385390 395 400 Ser Cys Ser Ser Phe Phe Gln Gln Phe Phe His Cys Tyr Cys ProVal 405 410 415 Gly Phe Gly Arg Arg Thr Asp Pro Asn Gly Asp Tyr Ile ArgArg Tyr 420 425 430 Leu Pro Val Leu Arg Gly Phe Pro Ala Lys Tyr Ile TyrAsp Pro Trp 435 440 445 Asn Ala Pro Glu Gly Ile Gln Lys Val Ala Lys CysLeu Ile Gly Val 450 455 460 Asn Tyr Pro Lys Pro Met Val Asn His Ala GluAla Ser Arg Leu Asn 465 470 475 480 Ile Glu Arg Met Lys Gln Ile Tyr GlnGln Leu Ser Arg Tyr Arg Gly 485 490 495 Leu Gly Leu Leu Ala Ser Val ProSer Asn Pro Asn Gly Asn Gly Gly 500 505 510 Phe Met Gly Tyr Ser Ala GluAsn Ile Pro Gly Cys Ser Ser Ser Gly 515 520 525 Ser Cys Ser Gln Gly SerGly Ile Leu His Tyr Ala His Gly Asp Ser 530 535 540 Gln Gln Thr His LeuLeu Lys Gln Gly Arg Ser Ser Met Gly Thr Gly 545 550 555 560 Leu Ser GlyGly Lys Arg Pro Ser Gln Glu Glu Asp Thr Gln Ser Ile 565 570 575 Gly ProLys Val Gln Arg Gln Ser Thr Asn 580 585 4 472 PRT Escherichia coli 4 MetThr Thr His Leu Val Trp Phe Arg Gln Asp Leu Arg Leu His Asp 1 5 10 15Asn Leu Ala Ile Ala Ala Ala Cys Arg Asn Ser Ser Ala Arg Val Leu 20 25 30Ala Leu Tyr Ile Ala Thr Pro Arg Gln Trp Ala Thr His Asn Met Ser 35 40 45Pro Arg Gln Ala Glu Leu Ile Asn Ala Gln Ile Asn Gly Leu Gln Ile 50 55 60Ala Leu Ala Glu Lys Gly Ile Pro Leu Leu Phe Arg Glu Val Asp Asp 65 70 7580 Phe Val Ala Ser Val Glu Ile Val Lys Gln Val Cys Ala Glu Asn Ser 85 9095 Val Thr His Leu Phe Tyr Asn Tyr Gln Tyr Glu Val Asn Glu Arg Ala 100105 110 Arg Asp Val Glu Val Glu Arg Ala Leu Arg Asn Val Val Cys Glu Gly115 120 125 Phe Asp Asp Ser Val Ile Leu Pro Pro Gly Ala Val Met Thr GlyAsn 130 135 140 His Glu Met Tyr Lys Val Phe Thr Pro Phe Lys Asn Ala TrpLeu Lys 145 150 155 160 Arg Leu Arg Glu Gly Met Pro Glu Cys Val Ala AlaPro Lys Val Arg 165 170 175 Ser Ser Gly Ser Ile Glu Pro Ser Pro Ser IleThr Leu Asn Tyr Pro 180 185 190 Arg Gln Ser Phe Asp Thr Ala His Phe ProVal Glu Glu Lys Ala Ala 195 200 205 Ile Ala Gln Leu Arg Gln Phe Cys GlnAsn Gly Ala Gly Glu Tyr Glu 210 215 220 Gln Gln Arg Asp Phe Pro Ala ValGlu Gly Thr Ser Arg Leu Ser Ala 225 230 235 240 Ser Ile Ala Thr Gly GlyLeu Ser Pro Arg Gln Cys Leu His Arg Leu 245 250 255 Leu Ala Glu Gln ProGln Ala Leu Asp Gly Gly Ala Gly Ser Val Trp 260 265 270 Leu Asn Glu LeuIle Trp Arg Glu Phe Tyr Arg His Leu Ile Thr Tyr 275 280 285 His Pro SerLeu Cys Lys His Arg Pro Phe Ile Ala Trp Thr Asp Arg 290 295 300 Val GlnTrp Gln Ser Asn Pro Ala His Leu Gln Ala Trp Gln Glu Gly 305 310 315 320Lys Thr Gly Tyr Pro Ile Val Asp Ala Ala Met Arg Gln Leu Asn Ser 325 330335 Thr Gly Trp Met His Asn Arg Leu Arg Met Ile Thr Ala Ser Phe Leu 340345 350 Val Lys Asp Leu Leu Ile Asp Trp Arg Glu Gly Glu Arg Tyr Phe Met355 360 365 Ser Gln Ile Ile Asp Gly Asp Leu Ala Ala Asn Asn Gly Gly TrpGln 370 375 380 Trp Ala Ala Ser Thr Gly Thr Asp Ala Ala Pro Tyr Phe ArgIle Phe 385 390 395 400 Asn Pro Thr Thr Gln Gly Glu Lys Phe Asp His GluGly Glu Phe Ile 405 410 415 Arg Gln Trp Leu Pro Glu Leu Arg Asp Val ProGly Lys Val Val His 420 425 430 Glu Pro Trp Lys Trp Ala Gln Lys Ala GlyVal Thr Leu Asp Tyr Pro 435 440 445 Gln Pro Ile Val Glu His Lys Glu AlaArg Val Gln Thr Leu Ala Ala 450 455 460 Tyr Glu Ala Ala Arg Lys Gly Lys465 470 5 681 PRT Arabidopsis thaliana 5 Met Ser Gly Ser Val Ser Gly CysGly Ser Gly Gly Cys Ser Ile Val 1 5 10 15 Trp Phe Arg Arg Asp Leu ArgVal Glu Asp Asn Pro Ala Ile Ala Ala 20 25 30 Ala Val Arg Ala Gly Pro ValIle Ala Leu Phe Val Trp Ala Pro Glu 35 40 45 Glu Glu Gly His Tyr His ProGly Arg Val Ser Arg Trp Trp Leu Lys 50 55 60 Asn Ser Leu Ala Gln Leu AspSer Ser Leu Arg Ser Leu Gly Thr Cys 65 70 75 80 Leu Ile Thr Lys Arg SerThr Asp Ser Val Ala Ser Leu Leu Asp Val 85 90 95 Val Lys Ser Thr Gly AlaSer Gln Ile Phe Phe Asn His Leu Tyr Asp 100 105 110 Pro Leu Ser Leu ValArg Asp His Arg Ala Lys Asp Val Leu Thr Ala 115 120 125 Gln Gly Ile AlaVal Arg Ser Phe Asn Ala Asp Leu Leu Tyr Glu Pro 130 135 140 Trp Glu ValThr Asp Glu Leu Gly Arg Pro Phe Ser Met Phe Ala Ala 145 150 155 160 PheTrp Glu Arg Cys Leu Ser Met Pro Tyr Asp Pro Glu Ser Pro Leu 165 170 175Leu Pro Pro Lys Lys Ile Ile Ser Gly Asp Val Ser Lys Cys Val Ala 180 185190 Asp Pro Leu Val Phe Glu Asp Asp Ser Glu Lys Gly Ser Asn Ala Leu 195200 205 Leu Ala Arg Ala Trp Ser Pro Gly Trp Ser Asn Gly Asp Lys Ala Leu210 215 220 Thr Thr Phe Ile Asn Gly Pro Leu Leu Glu Tyr Ser Lys Asn ArgArg 225 230 235 240 Lys Ala Asp Ser Ala Thr Thr Ser Phe Leu Ser Pro HisLeu His Phe 245 250 255 Gly Glu Val Ser Val Arg Lys Val Phe His Leu ValArg Ile Lys Gln 260 265 270 Val Ala Trp Ala Asn Glu Gly Asn Glu Ala GlyGlu Glu Ser Val Asn 275 280 285 Leu Phe Leu Lys Ser Ile Gly Leu Arg GluTyr Ser Arg Tyr Ile Ser 290 295 300 Phe Asn His Pro Tyr Ser His Glu ArgPro Leu Leu Gly His Leu Lys 305 310 315 320 Phe Phe Pro Trp Ala Val AspGlu Asn Tyr Phe Lys Ala Trp Arg Gln 325 330 335 Gly Arg Thr Gly Tyr ProLeu Val Asp Ala Gly Met Arg Glu Leu Trp 340 345 350 Ala Thr Leu Trp LeuHis Asp Arg Ile Arg Val Val Val Ser Ser Phe 355 360 365 Phe Val Lys ValLeu Gln Leu Pro Trp Arg Trp Gly Met Lys Tyr Phe 370 375 380 Trp Asp ThrLeu Leu Asp Ala Asp Leu Glu Ser Asp Ala Leu Gly Trp 385 390 395 400 GlnTyr Ile Thr Gly Thr Leu Pro Asp Ser Arg Glu Phe Asp Arg Ile 405 410 415Asp Asn Pro Gln Phe Glu Gly Tyr Lys Phe Asp Pro Asn Gly Glu Tyr 420 425430 Val Arg Arg Trp Leu Pro Glu Leu Ser Arg Leu Pro Thr Asp Trp Ile 435440 445 His His Pro Trp Asn Ala Pro Glu Ser Val Leu Gln Ala Ala Gly Ile450 455 460 Glu Leu Gly Ser Asn Tyr Pro Leu Pro Ile Val Gly Leu Asp GluAla 465 470 475 480 Lys Ala Arg Leu His Glu Ala Leu Ser Gln Met Trp GlnLeu Glu Ala 485 490 495 Ala Ser Arg Ala Ala Ile Glu Asn Gly Ser Glu GluGly Leu Gly Asp 500 505 510 Ser Ala Glu Val Glu Glu Ala Pro Ile Glu PhePro Arg Asp Ile Thr 515 520 525 Met Glu Glu Thr Glu Pro Thr Arg Leu AsnPro Asn Arg Arg Tyr Glu 530 535 540 Asp Gln Met Val Pro Ser Ile Thr SerSer Leu Ile Arg Pro Glu Glu 545 550 555 560 Asp Glu Glu Ser Ser Leu AsnLeu Arg Asn Ser Val Gly Asp Ser Arg 565 570 575 Ala Glu Val Pro Arg AsnMet Val Asn Thr Asn Gln Ala Gln Gln Arg 580 585 590 Arg Ala Glu Pro AlaSer Asn Gln Val Thr Ala Met Ile Pro Glu Phe 595 600 605 Asn Ile Arg IleVal Ala Glu Ser Thr Glu Asp Ser Thr Ala Glu Ser 610 615 620 Ser Ser SerGly Arg Arg Glu Arg Ser Gly Gly Ile Val Pro Glu Trp 625 630 635 640 SerPro Gly Tyr Ser Glu Gln Phe Pro Ser Glu Glu Asn Arg Ile Gly 645 650 655Gly Gly Ser Thr Thr Ser Ser Tyr Leu Gln Asn His His Glu Ile Leu 660 665670 Asn Trp Arg Arg Leu Ser Gln Thr Gly 675 680 6 540 PRT Drosophilamelanogaster 6 Met Asp Ser Gln Arg Ser Thr Leu Val His Trp Phe Arg LysGly Leu 1 5 10 15 Arg Leu His Asp Asn Pro Ala Leu Ser His Ile Phe ThrAla Ala Asn 20 25 30 Ala Ala Pro Gly Lys Tyr Phe Val Arg Pro Ile Phe IleLeu Asp Pro 35 40 45 Gly Ile Leu Asp Trp Met Gln Val Gly Ala Asn Arg TrpArg Phe Leu 50 55 60 Gln Gln Thr Leu Glu Asp Leu Asp Asn Gln Leu Arg LysLeu Asn Ser 65 70 75 80 Arg Leu Phe Val Val Arg Gly Lys Pro Ala Glu ValPhe Pro Arg Ile 85 90 95 Phe Lys Ser Trp Arg Val Glu Met Leu Thr Phe GluThr Asp Ile Glu 100 105 110 Pro Tyr Ser Val Thr Arg Asp Ala Ala Val GlnLys Leu Ala Lys Ala 115 120 125 Glu Gly Val Arg Val Glu Thr His Cys SerHis Thr Ile Tyr Asn Pro 130 135 140 Glu Leu Val Lys Ala Lys Asn Leu GlyLys Ala Pro Ile Thr Tyr Gln 145 150 155 160 Lys Phe Leu Gly Ile Val GluGln Leu Lys Val Pro Lys Val Leu Gly 165 170 175 Val Pro Glu Lys Leu LysLys Met Pro Thr Pro Pro Lys Asp Glu Val 180 185 190 Glu Gln Lys Asp SerAla Ala Tyr Asp Cys Pro Thr Ile Lys Gln Leu 195 200 205 Val Lys Arg ProGlu Glu Leu Gly Pro Asn Lys Phe Pro Gly Gly Glu 210 215 220 Thr Glu AlaLeu Arg Arg Met Glu Glu Ser Leu Lys Asp Glu Ile Trp 225 230 235 240 ValAla Arg Phe Glu Lys Pro Asn Thr Ala Pro Asn Ser Leu Glu Pro 245 250 255Ser Thr Thr Val Leu Ser Pro Tyr Leu Lys Phe Gly Cys Leu Ser Ala 260 265270 Arg Leu Phe Asn Gln Lys Leu Lys Glu Ile Ile Lys Arg Gln Pro Lys 275280 285 His Ser Gln Pro Pro Val Ser Leu Ile Gly Gln Leu Met Trp Arg Glu290 295 300 Phe Tyr Tyr Thr Val Ala Ala Ala Glu Pro Asn Phe Asp Arg MetLeu 305 310 315 320 Gly Asn Val Tyr Cys Met Gln Ile Pro Trp Gln Glu HisPro Asp His 325 330 335 Leu Glu Ala Trp Thr His Gly Arg Thr Gly Tyr ProPhe Ile Asp Ala 340 345 350 Ile Met Arg Gln Leu Arg Gln Glu Gly Trp IleHis His Leu Ala Arg 355 360 365 His Ala Val Ala Cys Phe Leu Thr Arg GlyAsp Leu Trp Ile Ser Trp 370 375 380 Glu Glu Gly Gln Arg Val Phe Glu GlnLeu Leu Leu Asp Gln Asp Trp 385 390 395 400 Ala Leu Asn Ala Gly Asn TrpMet Trp Leu Ser Ala Ser Ala Phe Phe 405 410 415 His Gln Tyr Phe Arg ValTyr Ser Pro Val Ala Phe Gly Lys Lys Thr 420 425 430 Asp Pro Gln Gly HisTyr Ile Arg Lys Tyr Val Pro Glu Leu Ser Lys 435 440 445 Tyr Pro Ala ThrCys Ile Tyr Glu Pro Trp Lys Ala Ser Leu Val Asp 450 455 460 Gln Arg AlaTyr Gly Cys Val Leu Gly Thr Asp Tyr Pro His Arg Ile 465 470 475 480 ValLys His Glu Val Val His Lys Glu Asn Ile Lys Arg Met Gly Ala 485 490 495Ala Tyr Lys Val Asn Arg Glu Val Arg Thr Gly Lys Glu Glu Glu Ser 500 505510 Ser Phe Glu Glu Lys Ser Glu Thr Ser Thr Ser Gly Lys Arg Lys Val 515520 525 Arg Arg Ala Thr Gly Ser Ala Pro Lys Arg Lys Arg 530 535 540 7 27DNA Homo sapiens misc_feature (1)..(27) primer 7 gggctctgcc acagggtgactgaggtc 27 8 18 DNA Homo sapiens misc_feature (1)..(18) primer 8aatacccgga ccccgctc 18 9 18 DNA Homo sapiens misc_feature (1)..(18)primer 9 caggtcccac aggcggta 18 10 27 DNA Artificial Synthetic primer 10cgcgaattcc tccctggagg agctggg 27 11 27 DNA Artificial Synthetic primer11 gcgagatctt caggcatcct tgctcgg 27 12 27 DNA Artificial Syntheticprimer 12 gcggatatcg cggcagctgt ggccccg 27 13 27 DNA ArtificialSynthetic primer 13 gcgaagcttt caggcatcct tgctcgg 27 14 37 DNAArtificial Synthetic primer 14 gcgagatctc cgccatcatg gcggcaactg tggcaac37 15 27 DNA Artificial Synthetic primer 15 gcgtctagat caggcatccttgctcgg 27 16 31 DNA Artificial Synthetic primer 16 gcgctcgagtcaggcatcct tgctcggcca g 31 17 334 DNA Homo sapiens misc_feature(308)..(308) n is a, c, g, or t 17 ctgcagtgct ttcttccagc agttcttccactgctactgc cctgtgggct ttggccgtcg 60 cacggacccc agtggggact acatcaggcgatacctgccc aaattgaaag cgttcccctc 120 tcgatacatc tatgagccct ggaatgccccagagtcaatt cagaaggcag ccaagtgcat 180 cattggtgtg gactacccac ggcccatcgtcaaccatgcc gagaccagcc ggcttaacat 240 tgaacgaatg aagcagattt accagcagctttcgcgctac cggggacttt tgtctaatgg 300 catctgtncc ttcctgtntg gaagactcagtcaa 334 18 302 DNA Homo sapiens misc_feature (139)..(139) n is a, c, g,or t 18 gcttacatac aagcgctttc aggccatcat cagccgcatg gagctgcccaagaagccagt 60 gggcttggtg accagccagc agatggagag ctgcagggcc gaggatccaggagaaccacg 120 acgagaccta cggcgtgcnc tccctggtag nagctggggt tccccactgtaaggacttgg 180 tcnagctgtn tggccaggag gtagagacag aagctctggc ccgcctggataagcacttng 240 gaanggaang nctgggttgc caactatgag agancccgaa tgaacgccaacttccctcct 300 gg 302 19 495 DNA Homo sapiens misc_feature (10)..(10) nis a, c, g, or t 19 aattcggcan gaggtgcctt atagagtcca actgttgtacgtatgtnacc ttcactagaa 60 atgtcccatc atcgtgggag gggagcaggg cacaggggatggtgtgcatt tagagcattg 120 ggttnggggc ttccctgttc cctcagcccc agtngagaggnaangagaat cggggccact 180 nncagaaaga gagtcaagca aacctgggna gggcaaatntntggagtggg aaggccaaag 240 gcccggggcc cagatttagt attnantagc agcgccttcggggtagcang gtggattcct 300 tttcctgnct gtntgntgnt ggnttctttt tccctnaggttanangtttg gcangaccaa 360 ctttcggnnt aattttttcc ancannctta ggcatggcannccntttncc cngttcaact 420 tntccaatgg gggttcaggn nattgcaatt cttggnggccaannggggag ggcnaggtta 480 gagagggtat ttncc 495 20 317 DNA Homo sapiensmisc_feature (19)..(19) n is a, c, g, or t 20 aaaattctct gctactggnctcagtcccag gcccttcctt gggcttntgg gactgagcat 60 gaggccatag acagatctaaaaagtttcca ccaccctaca gaagtacaca cagatacctg 120 actggtgtgg ggtatgcctgggtactgtaa taggagnnta agacagcaca cctacctttt 180 caggnnttta ggaacctaaaaattagaaag gggaattccc agctgtcaat tgntccttcc 240 ccagaagcta agaggccagccttcagaggc tacccaggga gctgtgaagg ggcaaggngt 300 caaacctgac ttcaatt 31721 334 DNA Homo sapiens misc_feature (308)..(308) n is a, c, g, or t 21ctgcagtgct ttcttccagc agttcttcca ctgctactgc cctgtgggct ttggccgtcg 60cacggacccc agtggggact acatcaggcg atacctgccc aaattgaaag cgttcccctc 120tcgatacatc tatgagccct ggaatgcccc agagtcaatt cagaaggcag ccaagtgcat 180cattggtgtg gactacccac ggcccatcgt caaccatgcc gagaccagcc ggcttaacat 240tgaacgaatg aagcagattt accagcagct ttcgcgctac cggggacttt tgtctaatgg 300catctgtncc ttcctgtntg gaagactcag tcaa 334 22 210 DNA Homo sapiensmisc_feature (31)..(31) n is a, c, g, or t 22 caggagggct ggttccaccacctggcccgg natgccgtgg cctgcttcct gacccgcggg 60 gacctntggn tcagctgggagagcggggtc cgggtattta atgagctgct cctggatgca 120 gatttaagcg tgaacgcaggcagctggatg tggctgtcct gcagtgcttt tttccagcag 180 ttnttccact gctactgccctgtgggtttt 210

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding the amino acid sequence at position −22 to 571 in SEQID NO:2; (b) a nucleotide sequence encoding the amino acid sequence atposition −21 to 571 in SEQ ID NO:2; (c) a nucleotide sequence encodingthe amino acid sequence at position 1 to 571 in SEQ ID NO:2; (d) anucleotide sequence encoding the amino acid sequence at position 191 to571 in SEQ ID NO:2; (e) a nucleotide sequence encoding the amino acidsequence at 1 to 492 in SEQ ID NO:2; (f) a nucleotide sequence encodingthe amino acid sequence at 493 to 505 in SEQ ID NO:2; (g) a nucleotidesequence encoding the amino acid sequence at 506 to 571 in SEQ ID NO:2;(h) a nucleotide sequence encoding the hCRY2 receptor having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97769; (i) a nucleotide sequence encoding the mature hCRY2receptor having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97769; and (j) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i).
 2. An isolated nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a), (b), (c), (d), (e),(f), (g), (h), or (i) of claim 1 wherein said polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.
 3. An isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a hCRY2 receptor having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g), (h), or (i) of claim 1.4. The isolated nucleic acid molecule of claim 3, which encodes anepitope-bearing portion of a hCRY2 receptor selected from the groupconsisting of: a polypeptide comprising amino acid residues from about 8to about 17 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 23 to about 30 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 55 to about 70 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 74 to about 81 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 136 to about 147in SEQ ID NO:2; a polypeptide comprising amino acid residues from about156 to about 165 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 231 to about 239 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 271 to about 279 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 298 toabout 309 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 316 to about 324 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 328 to about 334 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 415 to about 426in SEQ ID NO:2; and a polypeptide comprising amino acid residues fromabout 512 to about 519 in SEQ ID NO:2.
 5. An isolated nucleic acidmolecule, comprising a polynucleotide having a sequence selected fromthe group consisting of: (a) the nucleotide sequence of a fragment ofthe sequence shown in SEQ ID NO:1, wherein said fragment comprises atleast 50 contiguous nucleotides of SEQ ID NO:1, provided that saidfragment is not SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, or SEQ ID NO:22 or any subfragment thereof; and (b) anucleotide sequence complementary to a nucleotide sequence in (a).
 6. Amethod for making a recombinant vector comprising inserting an isolatednucleic acid molecule of claim 1 into a vector.
 7. A recombinant vectorproduced by the method of claim
 6. 8. A method of making a recombinanthost cell comprising introducing the recombinant vector of claim 7 intoa host cell.
 9. A recombinant host cell produced by the method of claim8.
 10. A recombinant method for producing an hCRY2 polypeptide,comprising culturing the recombinant host cell of claim 9 underconditions such that said polypeptide is expressed and recovering saidpolypeptide.
 11. An isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence selected from the groupconsisting of: (a) amino acids −22 to 571 in SEQ ID NO:2; (b) aminoacids −21 to 571 in SEQ ID NO:2; (c) amino acids 1 to 571 in SEQ IDNO:2; (d) amino acids 191 to 571 in SEQ ID NO:2; (e) amino acids 1 to492 in SEQ ID NO:2; (f) amino acids 493 to 505 in SEQ ID NO:2; (g) aminoacids 506 to 571 in SEQ ID NO:2; (h) the amino acid sequence of thehCRY2 polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97769; (i) the amino acidsequence of the mature hCRY2 polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No.97769; and (j)the amino acid sequence of an epitope-bearing portion of any one of thepolypeptides of (a), (b), (c), (d), (e), (f), (g), (h), or (i).
 12. Anisolated polypeptide comprising an epitope-bearing portion of the hCRY2receptor protein, wherein said portion is selected from the groupconsisting of: a polypeptide comprising amino acid residues from about 8to about 17 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 23 to about 30 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 55 to about 70 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 74 to about 81 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 136 to about 147in SEQ ID NO:2; a polypeptide comprising amino acid residues from about156 to about 165 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 231 to about 239 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 271 to about 279 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 298 toabout 309 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 316 to about 324 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 328 to about 334 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 415 to about 426in SEQ ID NO:2; and a polypeptide comprising amino acid residues fromabout 512 to about 519 in SEQ ID NO:2.
 13. The isolated polypeptide ofclaim 11, which is produced or contained in a recombinant host cell. 14.The isolated polypeptide of claim 13, wherein said recombinant host cellis mammalian.
 15. An isolated nucleic acid molecule comprising apolynucleotide encoding a hCRY2 polypeptide wherein, except for one tofifty conservative amino acid substitutions, said polypeptide has asequence selected from the group consisting of: (a) a nucleotidesequence encoding the amino acid sequence at position −22 to 571 in SEQID NO:2; (b) a nucleotide sequence encoding the amino acid sequence atposition −21 to 571 in SEQ ID NO:2; (c) a nucleotide sequence encodingthe amino acid sequence at position 1 to 571 in SEQ ID NO:2; (d) anucleotide sequence encoding the amino acid sequence at position 191 to571 in SEQ ID NO:2; (e) a nucleotide sequence encoding the amino acidsequence at 1 to 492 in SEQ ID NO:2; (f) a nucleotide sequence encodingthe amino acid sequence at 493 to 505 of SEQ ID NO:2; (g) a nucleotidesequence encoding the amino acid sequence at 506 to 571 of SEQ ID NO:2;(h) a nucleotide sequence encoding the hCRY2 receptor having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97769; (i) a nucleotide sequence encoding the mature hCRY2receptor having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97769; and (j) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i).
 16. An isolated hCRY2 polypeptide wherein,except for one to fifty conservative amino acid substitutions, saidpolypeptide has a sequence selected from the group consisting of: (a)amino acids −22 to 571 in SEQ ID NO:2; (b) amino acids −21 to 571 in SEQID NO:2; (c) amino acids 1 to 571 in SEQ ID NO:2; (d) amino acids 191 to571 in SEQ ID NO:2; (e) amino acids 1 to 492 in SEQ ID NO:2; (f) aminoacids 493 to 505 in SEQ ID NO:2; (g) amino acids 506 to 571 in SEQ IDNO:2; (h) the amino acid sequence of the hCRY2 polypeptide having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97769; (i) the amino acid sequence of the mature hCRY2polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No.97769; and (j) the amino acid sequence ofan epitope-bearing portion of any one of the polypeptides of (a), (b),(c), (d), (e), (f), (g), (h), or (i).
 17. An isolated antibody orportion thereof that specifically binds to the polypeptide of claim 11.18. An isolated antibody or portion thereof that specifically binds tothe polypeptide of claim 12.